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Chapter 4 Glycosylation Precursors

This chapter explores the intricate processes of glycosylation, focusing on the activation of monosaccharides into nucleotide sugars, which serve as high-energy donors. Different types of sugar transporters, including energy-independent and energy-dependent transporters, are discussed, highlighting their roles in glucose and other monosaccharide transport. The chapter further examines the intracellular sources of monosaccharides, the activation pathways, and the diverse functions of glycosyltransferases and other glycan-processing enzymes essential for glycan biosynthesis and modification.

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Chapter 4 Glycosylation Precursors

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  1. Chapter 4 Glycosylation Precursors Ya-Min Chi

  2. Intro • Glycosylation requires activation of monosaccharides to nucleotide sugar. • Nucleotide sugar is the high-energy donor form.

  3. Activated Form

  4. Glucose Transporters • Three types • Energy-independent diffusion transporter • e.g. Hexose transporter • Energy-dependent transporter • e.g. Sodium-dependent glucose transporter (SGLT) • Transporter that couple ATP phosphorylation with sugar import. • e.g. found in bacateria

  5. Mannose & Fucose Transport • Mannose • Energy-dependent transporter • Near border of enterocytes • Surface of kidney tubule epithelial • Energy-independent facilitated diffusion • Surface of many mammalian cells • Fucose • Found in several type of mammalian cells • Km-250µM

  6. Intracellular Source of Monosaccharides • Salvage • from glycoconjugates • Degradation at low pH in the lysosomes • Lysosomal carriers • Neutral sugar carrier • Km-50-75mM • N-acetylhexosamine carrier • Km-4mM • Acidic sugar carrier • Km-300-550µM

  7. Activation and Interconversion • Glycogen • Glycogenin • Glycogen phosphorylase • From UDP-Glc • Glucose • Building block of other sugar • Glycogen • Glucosylceramide • Dolichol-P-glucose

  8. Glucuronic Acid • UDP-GlcA • Synthesized from UDP-GlC • For • GAG biosynthesis • Some N-/O-linked glycans and glycosphingolipids • Iduronic Acid • C-5 epimer of glucuronic acid • Found in GAGs, dermatan sulfate, heparan sulfate, and heparin • Created from epimerization of GlcA

  9. Xylose • UDP-Xyl • Created from decarboxylation of UDP-GlcA • Initiate GAG synthesis • Mannose • Use for multiple types of glycan • GDP-Man • Primary activated donor • form Lipid-linked oligosaccharide on ER • Form dolichol-P-mannose in the ER membrane • Fucose • GDP-Fuc • From GDP-Man or directly from fucose

  10. Galactose • UDP-Gal • From Gal or UDP-Glc • N-Acetlyglucosamine • UDP-GlcNAc • From fructose or GlcNAc • N-Acetlygalactosamine • UDP-GalNAc • From GalNac or epimerization of UDP-GlcNAc • Sialic Acids • CMP-Sia • Many ways to obtain • Mostly occur in Golgi

  11. Bacteria and Plant • Animal • Fucose • The only deoxyhexoes in animal cell glycan • Bacteria and Plant • Varieties of sugars • Deoxysugars • From oxidation of C-4 • Deoxyaminosugars • Amino sugar- from keto sugar with addition of amino group from glutamine • Branched-chain sugars

  12. Nucleotide Sugar Transporters • Activated sugar must be transport • Needed in ER and Golgi • Negatively charged • Antiporters • Energy-independent • Organelle specific • Location correspond to glycosyltranferases’ location • Km-1-10µM • Export nucleoside monophosphates

  13. Control of Nucleotide Sugar Levels

  14. Other Modification

  15. Carrier Lipids • Transport sugar for glycosylation • e.g. • Undecaprenyl-P • Dolichol-P

  16. Chapter 5Glycosyltransferasesand Glycan-processing Enzymes

  17. Intro • Glycosyltransferases • Transport sugar from nucleotide sugar to substrate • Variety of enzymes • Glycosidase • Enzyme that break down glycosidic bond • For the formation of N-glycans • Other enzymes • e.g. sulfotransferases

  18. Glycosyltransferase Specificity • “one enzyme-one linkage” • Exception • some enzymes can produce the same linkage • e.g. human fucosyltransferases III-VII • Some have two separate active sites

  19. Protein/Glycoprotein Acceptors • Specificity of glycosylation reaction • Polypeptide chain of acceptor • Folding of protein acceptor

  20. GlycosyltransferaseSequence Families • Represent 1-2% of the genome • 30,000+ sequence • 90 families • 29 families’ structures have been determined

  21. Folding type • Two major types of of folding • GT-A • One single domain • Contain Rossmann fold • Contain DXD motif (asp-any residue-asp) • Metal-ion dependent • GT-B • Two domains • One is carboxy-termminal • Metal-ion independent

  22. Catalytic Mechanisms • Through either inversion or retention of stereochemistry at the anomeric carbon. inversion

  23. Kinetic Mechanisms • Bi Bi sequential kinetic mechanism • Donor substrate is bind first • Acceptor is release first

  24. Other Enzymes • Sulfotransferases • Cytoplasm and the Golgi • For glycosaminylglycans • Embryological development and physiology • For L-selectin ligand • Trafficking of lymphocytes • Use PAPS as sulfate donor

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